Previous attempts to develop a vaccine that would prevent disease associated with RSV infection have failed. In particular, a formalin treated, whole virus vaccine, known as the Lot 100 Pfizer vaccine, not only failed to protect but also produced enhanced disease when immunized children subsequently underwent natural infection with RSV. The exact cause of the lack of protective immunity and the potentiated disease are not completely understood. Serological markers that could predict the ability of a vaccine to protect against infection or identify the potential for enhanced disease would provide a safety test for the regulation of RSV vaccines. Investigators in other laboratories have suggested that there was an imbalance in the immune response following immunization that resulted in high titers of RSV specific binding antibody but little antibody that could neutralize virus or inhibit fusion of infected cells. Previous work in this laboratory demonstrated in an ELISA assay that formalin treated virus lost reactivity with F specific neutralizing MAbs but maintained or had enhanced reactivity with non-neutralizing F and G MAbs. In order to extend these findings, we have biotinylated 25 neutralizing and non-neutralizing F MAbs and 8 G MAbs that identify a minimum of 6 and 2 antigenic sites on the F and G glycoproteins, respectively. These biotinylated monoclonal antibodies were also used to develop an antigenic site blocking assay that can detect variations in the immune response to specific antigenic sites on the viral glycoproteins. During the coming year we will compare the antibody response of cotton rats infected intranasally with RSV to the response of animals immunized with a formalin inactivated RSV vaccine known to be associated with enhanced disease. The antigenic site blocking assay will also be used to evaluate the immune response of the children who received the Lot 100 vaccine. These studies will be done in collaboration with Dr. Mark Connors and Dr. Brian Murphy, LID, NIAID, who have the archived collection of animal and human sera.